1. Field of the Invention
The present invention relates generally to molecule separation techniques, and more specifically, to a method and apparatus for rapidly separating molecules, such as genomic DNA, using electric fields and flow distributions.
2. Related Art
The control of electric fields over large areas in fluidic systems is crucial for the accurate delivery and manipulation of biologically important molecules, such as DNA. Most biologically important molecules, including proteins, are electrically charged. Previous attempts at controlling such molecules involved manipulating same on a microscopic scale using electric fields. For example, in a method known as electrophoresis, charged molecules are migrated through a fluid under the influence of an applied electric field. In pulsed-field gel electrophoresis (“PFGE”), DNA of different sizes can be separated by alternating between uniform fields in different directions across a two-dimensional area of typically 30 cm×30 cm.
In principle, one can use two pairs of electrodes to create tunable fields in a two-dimensional area, one pair for each field component (i.e., vertical or horizontal directions). However, the resulting field is highly distorted, because the electrodes perturb the fields generated thereby. In conventional PFGE systems, this problem is solved by a method that uses many electrodes to clamp the electric potential along a closed contour, known as a contour-clamped homogenous electric field (“CHEF”) method. Fundamentally, this is equivalent to imposing a Dirichlet boundary condition to the Laplace equation governing the electric field. However, the CHEF method is inappropriate for fluidic applications where the array is only ˜1 cm×1 cm, because electrodes thereof can interfere with other functions of the array, such as sample loading and extraction. Further, such systems are not effective—even with the 24 electrodes typically used in commercial PFGE apparatuses, the field near the electrodes is not uniform. Additionally, microelectrodes inside fluidic channels are susceptible to erosion and bubble generation.
In the area of fluidic devices, and more particularly, in systems used in electrophoresis, it is desirable that the applied electric field in a layer of electrolyte be uniform. This is particularly true in traditional gel electrophoresis, which is used to assay proteins or nucleic acids, wherein many test samples are run simultaneously and/or in parallel. Non-uniformity of the electric field in a gel slab of such systems can cause a detrimental “smile effect,” which makes analysis of samples difficult and/or unreliable. Further, in PFGE, which can be used to fractionate large nucleic acids, not only does the electric field have to be homogenous, but the direction of the field must alternate with respect to the gel slab. Therefore, the ability to generate uniform electric fields in two-dimensional arrays, in addition to the ability to change the direction of such fields, is of paramount importance.
In CHEF systems, such as the system shown in FIG. 1a, complex electric fields having no divergence or curl can be generated in the layer of electrolyte using the plurality of electrodes to define an electric potential along a contour. Once the electric potential at each point of the boundary is set (i.e., at each of the electrodes), a Dirichlet boundary condition is established, with the electric potential Φ and the electric field E in the enclosed region of the electrolyte determined by the equations ∇2Φ=0 and E=−∇Φ when there is no current source (i.e., no electrode) inside the region.
Such an application, however, is not practical for small array applications, because different voltages must be applied to different locations and/or electrodes. Further, this approach requires numerous electrodes, electrolyte reservoirs, and complex driving circuits. Additionally, the method of FIG. 1a is inappropriate for micro-electrophoretic applications, because microfluidic devices are vulnerable to bubbles generated at the electrolyte/electrode interface inside the device.
Also in the area of fluidic devices, it is desirable to control the flow distribution of a layer of liquid contained therein. A common characteristic of such devices is that the Reynold's number of the fluid inside the device is so small that the flow is always laminar, i.e., non-turbulent. Also, because the layer of fluid is very thin, flow profiles that are usually parabolic can be ignored, and flow can be described in terms of its average flow velocity as a function of two position coordinates, for example, x and y. In addition, it can be assumed that the thickness of the fluid layer is so small that the overall shear force on each fluid element is dominated by the viscous shear between the fluid and the walls of the device. Viscous shear between any two fluid elements that are in different positions can be neglected. Therefore, the current J of the liquid flow is proportional to the negative gradient of the pressure P; that is, J=−σ∇P, wherein σ is the conductance tensor. Because liquid is incompressible, the current has no divergence, and the equation that describes the flow distribution is ∇2P=0.
In flow distribution systems presently used in the art, such as the system shown in FIG. 1b, a plurality of contact holes connected to exterior pressure regulators are provided near the perimeter of a region containing, for example, a fluid. This methodology allows for the control of flow distributions of the fluid, because Dirichlet boundary conditions thereof determine the solutions to Laplace equations. Such a system, however, is not practical for fluidic applications, because different pressures have to be applied to different locations, and numerous pressure sources are required.
What would be desirable, but has not yet been provided, is a technique that solves the above shortcomings while providing rapid separation of molecules. What would also be desirable, but has not yet been provided, is a method and apparatus for generating uniform electric fields and flow distributions for rapidly separating molecules.